Promoter sequence cloning and spatiotemporal expression pattern of rice amino acid permease gene OsAAP14

【Objective】 The objective of this study was to clone the promoter sequence of the rice amino acid permease gene OsAAP14 and analyze its spatiotemporal expression characteristics, which laid a foundation for the analysis of the biological function of the amino acid transport gene and provided a basis for understanding the response of rice to organic nitrogen sources and the absorption and utilization of amino acids.【Method】 The promoter sequence of the amino acid permease gene OsAAP14 was amplified in vitro by PCR technology, the promoter-GUS expression vector was constructed by pCAMBIA1391Z empty carrier plasmid, and the agrobacterium-mediated method was used to transform rice ZH11 callus, and the promoter-GUS transgenic plant of OsAAP14 gene was obtained, and the expression of specific cell sites of each tissue was observed by GUS staining of each tissue site and paraffin sectioning technology. The root GUS staining was car-ried out by five different amino acids treatments, and the gene expression corresponding to the above GUS staining site was further detected by combining real-time fluorescence quantification PCR technology, and the spatiotemporal expression characteristics of the gene were comprehensively analyzed.【Result】The OsAAP14 gene promoter sequence of 1993 bp was obtained by cloning and was consistent with the sequence of the reference sequence Nipponbare OsAAP14 (LOC_Os04g56470). This promoter sequence contained hormone or stress response elements such as MBS, P-box, ABRE and CGTCA-motif. Sixteen positive transgenic lines were identified from the promoter-GUS plants of OsAAP14 gene in T₀ generation. GUS staining and real-time fluorescence quantitative PCR results of different tissue parts of T1 generation materials showed that OsAAP14 gene was highly expressed in the basal parts of rice shoot elongation and leaves, and was also expressed in roots, leaf sheaths and panicles, but was least expressed in culms. Paraffin section analysis showed that OsAAP14 gene was highly expressed in root cortex parenchyma cells of roots, mesophyll cells of leaves, and inner cells of glumes of panicles. The expression of OsAAP14 gene in roots under histidine treatment in basic amino acids increased significantly(P<0.05, the same below) with increasing treatment time, and the relative expression of OsAAP14 gene in roots under gibberellin and abscisic acid treatments was also significantly increased.【Conclusion】The amino acid transport gene OsAAP14 expresses in all tissues of riceunder normal environment, and the expression is higher in the basal part and leave of rice. The expression of OsAAP14 in the root is strongly promoted by the exogenous amino acid and hormone treatments, indicating that the OsAAP14 gene may mainly participate in regulating the transportation of amino acids in the aboveground part of rice under normal environment, but it participates in regulating the transportation of rice root amino acidswhen the content of external amino acids and hormones increases.